Jurnal Malaria Tes

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ORIGINAL ARTICLE

Malaria detection with the Sysmex XE-2100 hematology

analyzer using pseudoeosinophilia and abnormal WBC

scattergram

Hee Jin Huh & Gwi Young Oh & Jung Won Huh &

Seok Lae Chae

Received: 2 January 2008 /Accepted: 8 March 2008 /Published online: 22 April 2008# Springer-Verlag 2008

Abstract Recent investigation using the Sysmex XE-2100

hematology analyzer (Sysmex Corporation, Japan) hasdemonstrated erroneously high eosinophil counts and

abnormal white blood cell (WBC) scattergrams in malaria

cases. This study was conducted to assess the diagnostic

efficiency of the Sysmex XE-2100 analyzer for malaria.

One hundred forty-four patients initially diagnosed with

Plasmodium vivax infection, 319 patients with febrile

illness, and 24 patients who underwent malaria treatment

were analyzed. Atypical features on Sysmex XE-2100

analyzer were categorized as pseudoeosinophilia (a gap of

more than 5% in eosinophil counts between the Sysmex

XE-2100 analyzer and microscopic examination) and

abnormal WBC scattergram. Pseudoeosinophilia or abnor-

mal WBC scattergram were detected in 100 of 144 malaria-

positive samples (sensitivity 69.4%, specificity 100%). The

samples with pseudoeosinophilia or abnormal WBC scatter-

grams showed significantly higher parasite counts than thesamples without pseudoeosinophilia or an abnormal WBC

scattergram ( P <0.05). All 24 samples from patients for

whom the malaria smear was repeated after malaria

treatment was initiated showed a normalized eosinophil

count and a normal WBC histogram. In conclusion,

attention to differential count and WBC scattergrams

provided by the Sysmex XE-2100 would be a valuable

tool in malaria detection.

Keywords Malaria . Pseudoeosinophilia . Abnormal WBC

scattergram . Sysmex XE-2100 hematology analyzer

Introduction

Malaria is diagnosed using a combination of clinical

observations, case history, and diagnostic tests, principally

microscopic examination of stained slides. However,

classical microscopic examination is labor intensive and

time consuming. Limitations of malaria diagnosis using this

method have led to the development of several new

techniques that simplify and speed up diagnosis and

increase sensitivity [1]. However, these all rely on clinical

suspicion and, consequently, a clinical request. Although

some methods lend themselves to automation (e.g., poly-

merase chain reaction) [2], no technique can yet be used for

routine clinical automated screening.

There is growing interest in the use of routine hemato-

logical blood analysis as a component of measurement for

the presumptive diagnosis of malaria infection [1, 3]. It has

been reported that abnormal depolarizing patterns detected

by a Cell-Dyn hematology analyzer (Abbott Diagnostics,

Santa, CA, USA) showed high sensitivity for malaria

Ann Hematol (2008) 87:755 – 759

DOI 10.1007/s00277-008-0486-8

H. J. Huh : S. L. Chae

Department of Laboratory Medicine, College of Medicine,

Dongguk University,

Goyang, Republic of Korea

G. Y. Oh

Department of Laboratory Medicine, Eone Reference Laboratory,

Seoul, Republic of Korea

J. W. Huh

Department of Laboratory Medicine, College of Medicine,

Ewha Womans University,

Seoul, Republic of Korea

S. L. Chae (*)

Department of Laboratory Medicine,

Dongguk University International Hospital,

Siksa-dong, Ilsandong-gu, Goyang-si,

Gyeonggi-do 411-773, Republic of Korea

e-mail: [email protected]



diagnosis [4]. One advantage of this method is its potential

to detect cases where clinical suspicion does not lead to a

request for a malaria test [5]. A few cases of malaria-

infected patients with pseudoeosinophilia on the Sysmex

XE-2100 hematology analyzer (Sysmex Corporation, Kobe,

Japan) were recently reported [6]. Moreover, abnormal

white blood cell (WBC) histograms were found in many

malaria-infected patients.The aim of this study was to determine the usefulness of

pseudoeosinophilia and abnormal WBC scattergrams, as

detected by the Sysmex XE-2100 analyzer, for the

diagnosis of malaria in a routine laboratory setting.

Materials and methods

The study group comprised a total of 487 samples from

patients for whom complete blood cell analysis and malaria

smears had been requested. Malaria patient group contained

144 patients that were initially diagnosed with Plasmodium

vivax infection (114 males and 30 females) and 24 patients

who underwent malaria treatment (18 males and six

females) at Dongguk University International Hospital and

Eone Reference Laboratory between June 2006 and

October 2007. Two hundred forty-two patients who

presented to the emergency department with a febrile

illness without malaria at Dongguk University International

Hospital between July 2007 and January 2008 and 77

patients diagnosed as bacteremia at Dongguk University

International Hospital November 2007 and January 2008

were included in control group. The diagnosis of 242

febrile patients were as followed: acute gastroenteritis 30

patients; respiratory tract infection 92 patients; urinary tract

infection 29 patients; other bacterial infection 36 patients;

fever due to other causes 55 patients. The diagnosis of

malaria was made by examining Wright-stained thick and

thin blood smears.

For complete blood cell analysis, we used the Sysmex

XE-2100 hematology analyzer. This instrument differen-

tiates WBCs using side fluorescence and side-scattered

light. An organic acid reagent binds specifically to the

granules of eosinophils and allows them to be discriminated

from neutrophils based on their higher side-scatter signal

intensities. WBC differential plots were examined for

abnormal WBC scattergrams. Manual WBC differential

counting was also performed by microscopic examination

of Wright-stained smears. Atypical features were catego-

rized as pseudoeosinophilia (a gap of more than 5% in

eosinophil counts between the Sysmex XE-2100 analyzer

and microscopic examination) and abnormal WBC scatter-

gram. The sensitivity and specificity of malaria detection

using pseudoeosinophilia and abnormal WBC scattergrams

were determined.

Parasitemia was indirectly calculated by assessing the

parasite numbers during the counting of 200 WBCs in the

thin blood film and, thereby, converting this to the number

of parasites per microliter of blood. Based on the parasite

number related with fever [7], samples from malaria

patients were subgrouped as a low parasitemia group

(<500 uL−1) and a high parasitemia group (≥500 uL−1).

Statistical analysis was performed using the StatisticalPackage for the Social Sciences (SPSS) program (version

11.0 for Windows; SPSS Inc., Chicago, IL, USA). Pearson

χ2 test and Fisher ’s exact test was used to compare

categorical variables. Student ’s t -test was used to compare

continuous variables between groups. P values of <0.05

were considered significant.

Results

Pseudoeosinophilia and abnormal WBC scattergrams

Of the 144 samples from malaria patients, 56 (38.9%)

showed pseudoeosinophilia. In samples with pseudoeosino-

philia, the mean (standard deviation (SD)) neutrophil count

by microscopic examination and Sysmex XE-2100 analyzer

were 67.1% (17.9) and 56.4% (15.1), respectively. The

mean (SD) eosinophil counts determined by microscopic

examination and Sysmex XE-2100 analyzer were 0.6%

(1.0) and 15.9% (9.5), respectively.

When compared to the normal WBC differential scatter-

gram (Fig. 1a), 75 samples from malaria patients showed

various abnormal features. The most strikingly noted

abnormal WBC scattergram was a nonclassified plot

extending from neutrophils toward the area of eosinophils

(n =33; Fig. 1 b). In these cases, neutrophils and eosinophil

counts are displayed as bars on the Sysmex XE-2100

analyzer. Other features encountered in malaria-positive

samples were two atypical eosinophil populations (n=31;

Fig. 1c), overlapping neutrophil and eosinophil populations

(n =7; Fig. 1d), and two neutrophil populations (n =4;

Fig. 1e). Among the 31 samples with two atypical

eosinophil populations, four samples simultaneously

showed two neutrophil populations (Fig. 1c).

Sensitivity and specificity

Using the abnormal WBC scattergrams for the initial

diagnosis, the sensitivity and specificity were 52.1% and

100%, respectively (Table 1). Using pseudoeosinophilia for

the initial diagnosis, the sensitivity and specificity were

38.9% and 100%, respectively. Overall, pseudoeosinophilia

or an abnormal WBC scattergram was detected in 100 of

144 malaria-positive samples (sensitivity 69.4%, specificity

100%).

756 Ann Hematol (2008) 87:755 – 759



According to Fisher ’s exact test, there was a significant

difference in frequency of pseudoeosinophilia or abnormal

WBC scattergram between malaria-positive samples and

control samples ( P <0.05).

Correlation of parasite count with pseudoeosinophilia

and abnormal WBC scattergram

The samples without pseudoeosinophilia or abnormal WBC

scattergrams showed significantly lower parasite counts

(517.9±866.3 uL−1 vs. 4,003.3±6,530.2 uL−1) and higher

pl at el et co un t (8 0, 97 7 ± 42 ,9 14 uL−1 v s. 5 4 ,6 00 ±

31,079 uL−1) than the samples with pseudoeosinophilia or

an abnormal WBC scattergram ( P <0.05; Table 2). Age,

sex, and WBC count were not different between the

samples with pseudoeosinophilia or an abnormal WBC

scattergram and the samples without pseudoeosinophilia or

an abnormal WBC scattergram ( P >0.05).

The sensitivity was 39.3% for the low parasitemia group

when pseudoeosinophilia or an abnormal WBC scattergram

was used for malaria detection, but this sensitivity increased

significantly, to 88.6%, for the high parasitemia group

(Table 3).

Follow-up results of malaria patients

During the treatment periods of 24 malaria patients, all cases

with an initial abnormal WBC scattergram and pseudoeosi-

nophilia were changed to normal. Among 24 samples, all

nine samples from patients who underwent treatment for less

than 3 days and had atypical features at initial diagnosis had a

normalized eosinophil count and WBC histogram.

a b c

ed

DIFF DIFF

DIFF DIFF

DIFF

Fig. 1 Abnormal WBC scatter-

grams generated by the Sysmex

XE-2100 hematology analyzer.

a Normal WBC differential

plots (blue neutrophil plots and

red eosinophil plots). b Non-

classified neutrophils and eosi-

nophils with the plot extending

from neutrophils toward the area

of eosinophils. c Two atypicaleosinophil populations and two

neutrophil populations. d Over-

lapping neutrophil and eosino-

phil populations. e Two

neutrophil populations

Table 1 Diagnostic efficiency

of the Sysmex-2100 analyzer

in malaria

Malaria Sensitivity

(%)

Specificity

(%) Negative

(n=319)

Positive

(n=144)

Pseudoeosinophilia 38.9 100

Positive 0 56

Negative 319 88

Abnormal WBC scattergram 52.1 100

Positive 0 75

Negative 319 69

Pseudoeosinophilia or abnormal WBC scattergram 69.4 100

Positive 0 100

Negative 319 44

Ann Hematol (2008) 87:755 – 759 757



Discussion

Automated blood cell analyzers can detect cases of malaria

in the absence of clinical suspicion [1, 8]. Suspicious plots

should be checked by stained blood film. The Cell-Dyn

3500 was the first autoanalyzer that allowed the detection

of malaria during routine investigation by complete bloodcell analysis [5]. The Cell-Dyn 3500 appears to detect

hemozoin-containing leukocytes according to their depola-

rizing properties [3]. It is believed that hemozoin-laden

monocytes, which have ingested parasites, are the major

component of the defining abnormal population identified

using the Cell-Dyn method [8, 9]. Hemozoin-containing

neutrophils seemed to be misidentified as eosinophils [9].

Moreover, it was suggested that the Cell-Dyn 3500 could

detect intraerythrocytic pigmented malarial parasites in rare

circumstances, such as relatively high parasitemia or

osmotic resistance [3].

Pseudoeosinophilia was detected in samples from ma-

laria patients using the Sysmex XE-2100 analyzer [6, 10].

The Sysmex XE-2100 analyzer cannot differentiate hemo-

zoin-containing monocytes from normal monocytes. How-

ever, the instrument detects hemozoin-containing

neutrophils and classifies them incorrectly as eosinophils

due to considerable side light scattering, which causes a

diagnosis of pseudoeosinophilia [6]. In this study, the mean

difference in neutrophil counts between microscopic exam-

ination and the Sysmex XE-2100 analyzer was 11%, and

the mean difference in eosinophil counts was 15%. This

difference could be explained by the incorrect classification

of neutrophils as eosinophils. Eleven samples with a normal

eosinophil count and an abnormal WBC scattergram

showed two atypical eosinophil populations or two neutro-

phil populations. This suggests that the instrument also

shows hemozoin-containing neutrophils to be abnormally

scattered eosinophil or neutrophil populations without

pseudoeosinophilia. The number of hemozoin-containing

neutrophils has been identified as an adverse prognosticindicator [11, 12]. This raises the possibility that the use of

the Sysmex XE-2100 analyzer for malaria diagnosis in our

study could also be useful prognostically.

The sensitivity of Cell-Dyn for the diagnosis of malaria

was variable, ranging from 43 – 95% [4, 8, 13, 14], and the

specificity was reported to be as high as 96 – 100%. This

study is the first evaluation of the sensitivity and specificity

of the Sysmex SE 2100 analyzer for malaria diagnosis. The

overall sensitivity and specificity were 69.4% (100/144)

and 100% (319/319), respectively, in our study. It has been

suggested that the kinetics of pigment-containing WBCs

may vary between different populations and could be

related to the severity of infection and host immunity

factors [4, 8, 13, 15]. In agreement with this, not all patients

in our study showed pseudoeosinophilia or abnormal WBC

scattergrams.

Although an abnormal WBC scattergram on the Sysmex

XE-2100 analyzer is specific for malaria diagnosis, it is

impossible to pay attention to all WBC histograms to detect

malaria in the laboratory. Among 100 samples with

pseudoeosinophilia or an abnormal WBC scattergram, 56

samples showed pseudoeosinophilia and 33 samples

showed blank eosinophil counts. Among the other 11

samples with a normal eosinophil count and an abnormal

Table 3 Comparison of pseudoeosinophilia and abnormal WBC scattergram between the low parasitemia group and the high parasitemia group

Low parasitemia group (n=56) High parasitemia group (n=88) P value

Pseudoeosinophilia (%) 17.8 (15/56) 34.1 (30/88) 0.5

Abnormal WBC scattergram (%) 17.8 (15/56) 68.2 (60/88) 0.000

Pseudoeosinophilia or abnormal WBC scattergram (%) 39.3 (22/56) 88.6 (78/88) 0.000

Table 2 Comparison of

parasite counts between

samples with and without

atypical features on the

Sysmex XE-2100 analyzer

Parasite count (Mean ± SD (uL−1)) P value

Pseudoeosinophilia

Positive (n=56) 2437.4±3452.2 0.002

Negative (n =88) 376.7±1378.6

Abnormal WBC scattergram

Positive (n=75) 4659.5±7246.5 0.000

Negative (n =69) 1067.5±2013.2

Pseudoeosinophilia or abnormal WBC scattergram

Positive (n=100) 4003.3±6530.2 0.000

Negative (n =44) 517.9±866.3

758 Ann Hematol (2008) 87:755 – 759



WBC scattergram, ten samples showed thrombocytopenia.

High incidence of thrombocytopenia has been reported in

malaria patients [16]. Disadvantage of detecting malaria

parasite by microscopic examination of stained slides is that

expertise and continuous training are required. Therefore,

the advantage of the Sysmex XE-2100 rather than micro-

scopic examination and Cell-Dyn is possible to suspect

malaria infection using high eosinophil count or uncountedeosinophil before review of slide or WBC scattergram.

After suspecting malaria infection in febrile patients due to

eosinophilia or a blank eosinophil count, careful review of

WBC histograms could be helpful in the diagnosis of

malaria. Interestingly, using pseudoeosinophilia and abnor-

mal WBC histograms, we detected several cases of malaria

during the recent 2 years without clinical suspicion.

This study demonstrated that samples with pseudoeosi-

nophilia or abnormal WBC scattergrams showed signifi-

cantly higher parasite counts than the samples without

pseudoeosinophilia or an abnormal WBC scattergram ( P <

0.05). Furthermore, the high parasitemia group showedhigher sensitivity for malaria diagnosis than the low

parasitemia group. These results are different from previous

studies using Cell-Dyn, which showed no correlation

between the amo unt of parasites and the abnormal

depolarizing pattern [4, 8, 13]. This difference could be

explained by the short half-life (6 – 7 hours) of neutrophils

compared with that of monocytes (2 – 3 weeks) [17].

Therefore, it is possible that recent parasites counts could

be reflected by the Sysmex XE-2100 abnormal WBC

scattergram.

In previous reports related to follow-up evaluation with

Cell-Dyn, samples of treated malaria with no residual

parasitemia showed abnormal depolarization [14, 18]. In

this study, pseudoeosinophilia and abnormal WBC scatter-

grams became normal within 3 days after the initiation of

treatment. The normalized eosinophil count and WBC

scattergram within a few days could be also explained by

the kinetics of hemozoin clearance, as the removal of

hemoglobin-containing neutrophils is faster than that of

hemoglobin-containing monocytes. If Sysmex XE-2100

analyzer is used as a clinical follow-up parameter for

eradication of malaria after treatment, further evaluation to

be applied to a larger cohort of patients is needed.

In conclusion, the Sysmex XE-2100 analyzer has an

advantage in that it is able to detect unexpected malaria

cases. Considering its low sensitivity, hematological anal-

ysis performed with this instrument should not be consid-

ered an alternative to existing methods for the diagnosis of

malaria. However, attention to differential counts and WBC

scattergrams provided by Sysmex XE-2100 may be

valuable in the diagnosis of malaria.

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Ann Hematol (2008) 87:755 – 759 759

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